Use of anti-connexin agents for enhancing the therapeutic effect of acetylcholinesterase inhibitors

ABSTRACT

This invention relates to improvements in therapeutic neurological and neuropsychic treatments using acetylcholinesterase inhibitors. More specifically, the invention enables the effects of the reversible acetylcholinesterase inhibitor donepezil to be potentiated by certain molecules, referred to here as connexin-blocking agents. Said connexin-blocking agent is preferably meclofenamic acid.

This invention relates to improvements in therapeutic neurological andneuropsychic treatments using acetylcholinesterase inhibitors. Morespecifically, the invention enables the effects of acetylcholinesteraseinhibitors to be potentiated by certain molecules, referred to here asconnexin-blocking agents.

Cognitive disorders are a category of mental health disorders thatprimarily affect learning, memory, perception, and problem solving, andinclude amnesia, dementia, and delirium. Causes vary between thedifferent types of disorders but most include damage to the memoryportions of the brain. Treatments depend on how the disorder is caused.Medication and therapies are the most common treatments; however, forsome types of disorders such as certain types of amnesia, treatments cansuppress the symptoms but there is currently no cure.

Enhancement of cognitive function occurs when the action ofAcetylcholine (Ach) is increased via inhibition of its metabolizingenzymes, principally the acetylcholinesterase enzyme (AChE).Accordingly, the strategy of increasing cholinergic activity to restorecognitive functions has been a primary and enduring therapeutic tactic.

Nowadays, several acetylcholinesterase inhibitors (ChEIs, or AChEIs oranti-cholinesterase agents) have been shown to reduce the rate at whichacetylcholine is broken down and hence increase its concentration in thebrain (thereby combating the loss of ACh caused by the death of thecholinergic neurons). Acetylcholinesterase inhibitors enhance neuronaltransmission by increasing the availability of ACh in muscarinic andnicotinic receptors. According to findings of some researchers, theseChEIs may have psychotropic effects and may play an important role incontrolling neuropsychiatric and behavioral disturbances in patientswith cognitive disorders. These agents may also contribute to themanagement of other disorders with cholinergic system abnormalities andneuropsychiatric symptoms such as visual hallucinations.

Donepezil is a well-known AChEI. It has been proposed for treatingnumerous cognitive disorders (Lewy body dementia, vascular dementia,sleep apnea, mild cognitive impairment, schizophrenia, CADASIL syndrome,attention deficit disorder, postcoronary bypass cognitive impairment,cognitive impairment associated with multiple sclerosis, and Downsyndrome). It has been approved for treating mild to moderateAlzheimer's disease.

Alzheimer's disease (AD) is an irreversible, progressive disorder inwhich brain cells (neurons) deteriorate, resulting in the loss ofcognitive functions, primarily memory, judgment and reasoning, movementcoordination, and pattern recognition. In advanced stages of thedisease, all memory and mental functioning may be lost. The death of thenerve cells occurs gradually over a period of years. It is associatedwith senile dementia which is the mental deterioration (loss ofintellectual ability) that is associated with old age. They arecurrently 5.3 million people with AD in the United States, and more thanhalf of these individuals will likely be categorized as having moderateor severe disease. These advanced stages of AD extend over a period ofseveral years and are often the most difficult for both patients andcaregivers. An important component of the pathophysiology of AD,recognized more than 30 years ago, is degeneration of the cholinergicsystem. Early histologic studies showing loss of cholinergic activity asAD progresses are supported by several modern lines of investigationusing advanced imaging techniques, including positron imaging techniques(PET) and magnetic resonance imaging (MRI). The cholinergicabnormalities seen in AD are not viewed as the cause of the disorder,but cholinergic involvement is significant because it is universal,correlates with cognitive defects, and is one of the fewpathophysiologic phenomena that can be addressed with currently approvedtreatment options.

However, the acetylcholinesterase-inhibitors that have been approved sofar (and in particular donepezil), have been less promisingtherapeutically as they produce only modest improvements in cognitivefunction and the induced cognitive gain (if any) only lasts few months.

There is therefore an urgent need of new treatments enabling to reduceand/or impair the loss of cognitive symptoms more efficiently and moredurably for all patients suffering from cognitive disorders, especiallythose suffering from Alzheimer in advanced stages.

The present invention fulfills this need by disclosing a new therapeuticproduct showing improved effects on memory loss than the existingtreatments.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the θ-hippocampal analysis of adult mice (4-5 month oldmice—6 first columns) and elderly mice (17-18 month-old mice—6 lastcolumns) after intraperitoneal administration of 0.1 mg/kg or 0.3 mg/kgof donepezil (DZP) associated or not with 1 mg/kg of meclofenamic acid(MFA).

FIG. 2 shows the total alternation of elderly mice (17-18 month-oldmice) after intraperitoneal administration of NaCl (vehicle) or of 0.1mg/kg, 0.3 mg/kg or 1 mg/kg of donepezil alone (DZP) or of 1 mg/kg ofmeclofenamic acid alone (MFA) or of the combination MFA and DZP (lastcolumn). Alternation has been recorded in a T-maze device.

FIG. 3 shows the alternation of “elderly” mice by blocks of twoconsecutive trials, after intraperitoneal administration of NaCl(vehicle), donepezil alone (0.1 mg/kg), MFA alone (1 mg/kg) and acombination of donepezil and MFA (last group of columns).

DESCRIPTION OF THE INVENTION

The present Inventors have hereafter demonstrated that the associationof Meclofenamic Acid (MFA) with an acetylcholinesterase inhibitor suchas donepezil surprisingly results in qualitatively superior preclinicalgains compared to higher doses of the acetylcholinesterase inhibitoralone.

Meclofenamic acid has been described as a “connexin-blocking” agenthaving a non-steroidal anti-inflammatory activity.

In the context of this invention, a “connexin-blocking” agent is achemical molecule, a protein, a protein fragment or a nucleic acid (forexample RNAi) capable of inhibiting the functional activity ofconnexins, directly and/or indirectly, and more generally any type ofintercellular junctions, and/or capable of functionally inhibiting,directly and/or indirectly any cellular activity involving aconnexin-type protein. Such an agent can also be referred to as an“anti-connexin molecule”.

Various molecules are known for blocking the gap junctions viaconnexins. Among them, the family of fenamates includes the followingcompounds: meclofenamic acid, mefenamic acid, flufenamic acid, niflumicacid and tolfenamic acid. These compounds all have a non-steroidalanti-inflammatory activity, but this activity is not responsible fortheir capacity to block the gap junctions. It has indeed been suggestedthat the fenamates instead establish a direct interaction with theconnexins or with the protein membrane interfaces that may influence theconformation of the connexins and therefore the functional role thereof(Harks E G, The Journal of Pharmacology and Experimental Therapeutics2001 September, 298(3): 1033-41).

Benzoic 2-[(2,6-di-chloro-3-phenyl)amino]acid, more commonly known asmeclofenamic acid (MFA), is a non-steroidal anti-inflammatory agent anda peripheral analgesic of the fenamate class, a prostaglandin inhibitor,described among the water-soluble blockers as being one of the mosteffective for reversibly blocking the gap junctions. In addition,meclofenamic acid is not specific to a type of connexin and is thereforeeffective for blocking a large number of cerebral connexins (Pan F, VisNeurosciences 2007, Jul.-Aug.; 24(4): 609-18).

Glycyrrhetinic acid derivatives refer to 18-β-glycyrrhetinic acid (BGA)also known as “enoxolone”, 18-α-glycyrrhetinic acid and carbenoxoloneacid, which are triterpinoid saponins known for inhibiting the11-hydroxysteroid dehydrogenase enzyme. Moreover, these compounds arecapable of very effectively inhibiting the gap junctions (Pan F, VisNeurosciences 2007, Jul.-Aug.; 24(4): 609-18).

Members of the quinine family, such as mefloquine (LARIAM), quinine andquinidine, also have a strong antagonist power on the gap junctions(Srinivas M, PNAS 2001, 98: 10942-10947; Pan F, Vis Neurosciences 2007,Jul.-Aug.; 24(4):609-18).

Some anesthetic agents, such as halothane, enflurane and isoflurane,have a rapid and reversible gap junction blocking effect (Burt J M, etal, Circ Research. 1989; 65: 829-37).

Moreover, oleamide (cis-9-octadecenamide), the first amide of oleicacid, also has an inhibiting action on the connexin molecules 43 and 32(Guan X. et al, J. Cell Biol 1997; 139: 1785-92).

In addition, cyclodextrins (α-cyclodextrin (α-CD), β-cyclodextrin((β-CD) and γ-cyclodextrin (γ-CD)), which are natural cyclicaloligosaccharides of α-D-glucopyranose, have proven anti-connexinproperties (Locke D. et al, J. Biol Chem 2004; 279: 22883-92).

Lipophilic agents and fatty acids such as oleic acid, palmitoleic acid,decaenoic acid and myristoleic acid, the PKC inhibitor staurosporine,cardiac glycosides such as strophanthidin and ouabin,delta-9-tetrahydrocannabinol, 2-aminoethoxydiphenyl borate acetic acidand propionic acid, 12-O-tetradecanoylphorbol-13-acetate (TPA), 2,3butandione monoxime, carbachol, noradrenaline, FGF-2, angiotensin-II,Atrial Natriuretic factor ANF, VEGF, 1-oleyl-2-acetyl-sn-glycerol,11,12-epoxyeicosatrienoic acid, lidocaine, tonabersat, Nexagon andPeptagon (from CoDa Therapeutics) have been also proposed as acutelyuncoupling cardiovascular gap junctions (Dhein S., CardiovascularResearch, 2004 (62) 287-298).

Finally, 2-aminoethyldiphenyl borate (2-APB) is a compound recentlyidentified as a gap junction-blocking agent (Bai D, J Pharmacol ExpTher, 2006 December; 319(3): 1452-8). This modulator of the inositol1,4,5-triphosphate receptor however fairly specifically targets certainconnexins, such as connexins 26, 30, 36, 40, 45 and 50 (Bai D, JPharmacol Exp Ther, 2006 December; 319(3): 1452-8).

Similarly, other molecules have recently been proposed for blocking theextracellular connexin domain—a domain that is important for thefunctioning of the gap junctions. It involves in particular antibodiesdirected against the extracellular connexin domain (Hofer A et al, Glia1998; 24: 141-54; Meyer R A, J. Cell Biol. 1992; 119: 179-89) or smallpeptides mimicking specific sequences conserved by the extracellularloops E1 and E2 of the connexins (Dahl G. et al, Biophys J, 1994; 67:1816-22); in particular, the peptides corresponding to the extracellularsequences include the conserved patterns QPG and SHVR of E1 (Gap26) andthe conserved pattern SRPTEK of E2 (Gap27) of the connexins are moreeffective for blocking the gap junctions (Chaytor A T et al, J. Physiol1997; 503: 99-110).

Moreover, the formation of functional gap junctions can be regulated bymeans of connexin phosphorylation. Indeed, phosphorylation of certainprotein domains of the hexamer sub-units leads to an inhibition in thefunctionality of the gap junctions, according to the phosphorylationsite, by closing the channels or by reducing the presence at themembrane (modification of traffic and half-life of sub-units) (Scemes E,Glia 2008 Jan. 15, 56(2): 145-53; Postma F R, J Cell Biol 1998 Mar. 9,140(5): 1199-209; Shaw R M, Cell 2007, Feb. 9, 128(3): 547-60; Fabrizi GM, Brain 2007 February, 130 (Pt2): 394-403).

Thus, molecules can have an indirect gap junction-blocking effect, viathe phosphorylation levels of the connexins. They are in particular:lysophosphatidic acid, thrombin and neuropeptides, such as endothelin(Postma F R, J Cell Biol 1998 Mar. 9, 140(5): 1199-209). In a preferredembodiment of the invention, the connexin-blocking agent has an indirecteffect on the connexins and the gap junctions, and it is chosen from thegroup consisting of: lysophosphatidic acid, thrombin and neuropeptides,such as endothelin.

In the context of this invention, the connexin-blocking agents areadvantageously chosen from: long-chain alcohols (for example, heptanoland octanol), fenamates (for example, meclofenamic acid, mefenamic acid,flufenamic acid, niflumic acid, tolfenamic acid), arylaminobenzoates,aminosulfonates (for example taurine), glycyrrhetinic acid derivatives(for example, 18-β-glycyrrhetinic acid, 18-α-glycyrrhetinic acid andcarbenoxolone), oleamides (for example, cis-9-octadecenamide), ortetraalkylammonium ions and polyamines (such as spermine andspermidine), quinine derivatives (such as mefloquine, quinine,quinidine), 2-ABP, anesthetic agents (halothane, enflurane orisoflurane), cyclodextrins (α-cyclodextrin (α-CD), β-cyclodextrin(β-CD), and γ-cyclodextrin (γ-CD)), antibodies directed against theextracellular domain of the connexins or peptides with conservedpatterns mimicking this particular domain (in particular Gap26 andGap27), oleic acid, palmitoleic acid, decaenoic acid, myristoleic acid,staurosporine, strophanthidin, ouabin, delta-9-tetrahydrocannabinol,2-aminoethoxydiphenyl borate acetic acid, propionic acid,12-O-tetradecanoylphorbol-13-acetate (TPA), 2,3 butandione monoxime,carbachol, noradrenaline, FGF-2, angiotensin-II, Atrial Natriureticfactor ANF, VEGF, 1-oleyl-2-acetyl-sn-glycerol,11,12-epoxyeicosatrienoic acid, lidocaine, tonabersat (SB-220453,(cis-(−)-6-acetyl-4S-(3-chloro-4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-3S-ol),Nexagon and Peptagon (from CoDa Therapeutics). These different moleculesare specifically described in the following articles: Srivinas M,Connexins: A Guide, Humana Press 2009, Chapter 8, pages 207-224;Srinivas M, Molecular Pharmacology 2003 June, 63(6): 1389-97; Harks E G,The Journal of Pharmacology and Experimental Therapeutics 2001September, 298(3): 1033-41; and Salameh A, Biochimica et Biophysica Acta1719 (2005) 36-58; Dhein S., Cardiovascular Research, 2004 (62) 287-298.

These compounds are provided as examples, and the invention relates toany molecule having the properties of functional blocking, direct orindirect, of the connexins or gap junctions.

Moreover, it should be noted that the anti-inflammatory molecules canindirectly produce, by their action on the prostaglandin synthase, astructural modification of the connexins (the regulation of the connexinexpression levels or of the phosphorylation thereof occurs in particularvia PI3K and PKA, themselves dependent on the activity levels of Cox, NOand PG synthetase, targets of anti-inflammatories). This modification,in the sense of a reduction in the presence of the connexins in thejunctions, indirectly causes a reduction in the functional activity ofthe connexins similar to a direct blocking of the connexins.Consequently, the use of these molecules will produce the desired effect(blocking of connexins) and is not an obstacle to combined use at a lowdose with psychotropic agents (Yao J, Morioka T & Oite T.: Kidney Int.2000; 57: 1915-26. Yao J, Hiramatsu N, Zhu Y, et al.: J Am Soc Nephrol.2005; 16: 58-67; Figueroa X F, Alvina K, Martinez A D, et al.: MicrovascRes. 2004; 68: 247-57 Alldredge B T.: J Clin Pathol. May 12 2008;Lai-Cheong J E, Arita K & McGrath J A.: J Invest Dermatol. 2007; 127:2713-25, and Giepmans B N.: Cardiovasc Res. 2004; 62: 233-45).

However, at the low doses involved in the present invention,anti-connexin agents such as meclofenamic acid have no effect on Cox, NOand PG synthetase, and exhibit only an anti-connexin activity which isindependent of these enzymes.

All anti-inflammatory molecules having a direct or indirectanti-connexin activity are encompassed in the present invention.

The present invention thus relates to a new combination productcontaining at least one connexin-blocking agent and anacetylcholinesterase inhibitor, as combination products forsimultaneous, separate or sequential use over time, in patientssuffering from cognitive disorders.

The connexin-blocking agent can advantageously improve the therapeuticeffect of acetylcholinesterase inhibitors prescribed by physicians fortreating a patient suffering from cognitive disorders.

The said connexin-blocking agent has been described above. In apreferred embodiment, it is chosen in the group including: meclofenamicacid, mefenamic acid, flufenamic acid, niflumic acid, tolfenamic acid,18-β-glycyrrhetinic acid also known as “enoxolone”, 18-α-glycyrrhetinicacid, carbenoxolone acid, mefloquine, quinine, quinidine, oleamide(cis-9-octadecenamide), oleic acid, palmitoleic acid, decaenoic acid,myristoleic acid, staurosporine, cyclodextrins (α-cyclodextrin (α-CD),β-cyclodextrin ((β-CD) and γ-cyclodextrin (γ-CD)), tonabersat(SB-220453,(cis-(−)-6-acetyl-4S-(3-chloro-4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-1enzopyran-3S-ol), Nexagon and Peptagon (from CoDa Therapeutics).

In a more preferred embodiment, it is chosen in the group including:meclofenamic acid, mefloquine, 18-β-glycyrrhetinic acid andcarbenoxolone.

In a rather preferred embodiment, said connexin-blocking agent ismeclofenamic acid (MFA).

An “acetylcholinesterase inhibitor” (often abbreviated as “AChEI”) or“anti-cholinesterase” is a chemical compound that inhibits thecholinesterase enzyme from breaking down acetylcholine, increasing boththe level and duration of action of the neurotransmitter acetylcholine.They occur naturally as venoms and poisons and are used medicinally totreat myasthenia gravis, glaucoma, Alzheimer's disease, Levy bodyDementia or as an antidote to anticholinergic poisoning.

The combination product of the invention differs from the prior art inthat it contains, as active principles, a cholinergic agent (which isnot an acetylcholinergic effector but an acetylcholinesteraseinhibitor), and a connexin-blocking agent. In a preferred embodiment,the combination product of the invention contains, as sole activeprinciples, said acetylcholinesterase inhibitor and saidconnexin-blocking agent. In particular, the combination productpreferably does not contain effective amount of a ketone body precursorsuch as medium chain triglycerides having 5-12 carbon chains. In apreferred embodiment, it does not contain effective amount ofphosphodiesterase 7 inhibitor (PDE7). In another preferred embodiment,it does not contain effective amount of axomadol. In another preferredembodiment, it does not contain effective amount of bupropion.

However, the composition can, in addition to the two active principles,comprise any pharmaceutical vehicle, stabilizer, adjuvant and the likeas frequently used in the art. Examples of pharmaceutically acceptablevehicles include (but are not limited to): water; aqueous vehicles suchas, but not limited to, sodium chloride solution, Ringer's solution,dextrose solution, dextrose and sodium chloride solution, and lactatedRinger's solution; water-miscible vehicles such as, but not limited to,ethyl alcohol, polyethylene glycol, and polypropylene glycol; andnonaqueous vehicles such as, but not limited to, corn oil, cottonseedoil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, andbenzyl benzoate. The skilled person well knows which vehicles can beused.

According to a preferred embodiment, this composition is formulated fororal administration (including buccal cavity or sublingually). Otherinteresting formulations include formulations for intraperitoneal (i.p),intravenous (i.v.), subcutaneous (s.c.), intramuscular (i.m.),transcutaneous, transdermal, intrathecal and intracranialadministrations. Still other formulations include epidural, submucosal,intranasal, ocular cul-de-sac and rectal routes of administration, aswell as administration by pulmonary inhalation. The skilled person wellknows which vehicles can be used in each kind of composition.

A variety of administration means, including but not limited tocapsules, tablets, syrups, creams and ointments, suppositories, patchesor any reservoir capable of containing and dispensing the two activeprinciples, can be used for formulating the above-describedcompositions. The skilled person well knows which vehicles can be usedin each case.

In a preferred embodiment, said acetylcholinesterase inhibitor is chosenfrom Acephate, Azinphos-methyl, Bensulide, Cadusafos, Chlorethoxyfos,Chlorfenvinphos, Chlorpyrifos, Chlorpyrifos-Methyl, Coumaphos,Cyclosarin, Demeton, Demeton-S-Methyl, Diazinon, Dichlorvos,Dicrotophos, Diisopropyl fluorophosphate (Guthion),Diisopropylphosphate, Dimethoate, Dioxathion, Disulfoton, EA-3148,Echothiophate, Ethion, Ethoprop, Fenamiphos, Fenitrothion, Fenthion,Fosthiazate, Isofluorophate, Isoxathion, Malaoxon, Malathion,Methamidophos, Methidathion, Metrifonate, Mevinphos, Monocrotophos,Naled, Novichok agent, Omethoate, Oxydemeton-Methyl, Paraoxon,Parathion, Parathion-Methyl, Phorate, Phosalone, Phosmet, Phostebupirim,Phoxim, Pirimiphos-Methyl, Sarin, Soman, Tabun, Temefos, Terbufos,Tetrachlorvinphos, Tribufos, Trichlorfon, Demecarium, Onchidal,acetylcholinesterase inhibitor chosen among: Aldicarb, Bendiocarb,Bufencarb, Carbaryl, Carbendazim, Carbetamide, Carbofuran, Chlorbufam,Chloropropham, Ethienocarb, Ethiofencarb, Fenobucarb, Fenoxycarb,Formetanate, Furadan, Ladostigil, Methiocarb, Methomyl, Miotine, Oxamyl,Phenmedipham, Pinmicarb, Pirimicarb, Propamocarb, Propham, Propoxur,Ganstigmine, Neostigmine, Phenserine and its enantiomer Posiphen,Physostigmine, Pyridostigmine, Rivastigmine, eptastigmine(heptylphysostigmine), Acotiamide, Ambenonium, Donepezil, Edrophonium,Galantamine, its derivatives SPH 1371, SPH 1373, SPH 1375 and SPH 1286((−)N-(3-piperidinopropyl)-N-demethylgalantamine), Huperzine A, itsprodrug ZT 1((5R,9R)-5-(r-chloro-2-hydroxy-3-methoxybenzylidene-amino)-11-ethuidene-7-methyl-1,2,5,6,9,10-hexahydro-5,9-methanocycloocta[b]pyridin-2-one),Minaprine, Tacrine, tolserine(3,4,8b-trimethyl-2,3α-dihydro-1H-pyrrolo[2,3-b]indol-7-yl)N-(2-methylphenyl)carbamate), Zanapezil, ER 127528(1-(3-fluorobenzyl)-4-[(2-fluoro-5,6-dimethoxy-1-indanone-2-yl)methyl]piperidinehydrochlo-ride), thiatolserine, RS 1259 (N,N-dimethylcarbamic acid 4-[1(S)-(methylamino)-3-(4 nitrophenoxy)propyl]phenyl ester hemifumarate),ipidacrine (NIK-247), velnacrine(9-Amino-1,2,3,4-tetrahydro-1-acridinol), zifrosilone(2,2,2-trifluoro-1-[3-(tri-methylsilyl)phenyl]ethanone), T 82(2-[2-(1-benzylpiperidin-4-yl)ethyl]-2,3-dihydro-9-methoxy-1H-pyrrolo[3,4-b]quinolin-1-onehemifumerate), CI 1002 (or PD 142676,1,3-dichloro-6,7,8,9,10,12-hexahydroazepino[2,1-b]-quinazoline), CHF2060 (N-heptylcarbamic acid2,4a,9-trimethyl-2,3,4,4a,9,9a-hexahydro-1,2-oxazino[6,5-b]indol-6-ylester-L-tartrate), MF 268(N-[8-(cis-2,6-dimethylmorpholin-4-yl)octyl]car-bamic acid(3aS,8aR)-1,3a,8-trimethyl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indol-5-ylester L-bitartrate hydrate), TV 3326(N-propargyl-3R-aminoindan-5-yl-ethyl methyl carbamate), Latrepirdine(Dimebolin),(−)-12-amino-3-chloro-9-ethyl-6,7,10,11-tetrahydro-7,11-methanocycloocta[b]quinolinehydrochloride (huperzine X), 3-(2-[1-(1,3-dioxolan-2-ylmethyl)piperidin-4-yl]ethyl)-3,4-dihydro-2H-1,3-benzoxazine-2,4-dionehydro-chloride (E 2030) and the pharmaceutically salts thereof.

Said acetylcholinesterase inhibitor can have reversible or irreversibleeffects.

In a more preferred embodiment, said acetylcholinesterase inhibitor isan irreversible acetylcholinesterase inhibitor chosen among: Acephate,Azinphos-methyl, Bensulide, Cadusafos, Chlorethoxyfos, Chlorfenvinphos,Chlorpyrifos, Chlorpyrifos-Methyl, Coumaphos, Cyclosarin, Demeton,Demeton-S-Methyl, Diazinon, Dichlorvos, Dicrotophos, Diisopropylfluorophosphate (Guthion), Diisopropylphosphate, Dimethoate, Dioxathion,Disulfoton, EA-3148, Echothiophate, Ethion, Ethoprop, Fenamiphos,Fenitrothion, Fenthion, Fosthiazate, Isofluorophate, Isoxathion,Malaoxon, Malathion, Methamidophos, Methidathion, Metrifonate,Mevinphos, Monocrotophos, Naled, Novichok agent, Omethoate,Oxydemeton-Methyl, Paraoxon, Parathion, Parathion-Methyl, Phorate,Phosalone, Phosmet, Phostebupirim, Phoxim, Pirimiphos-Methyl, Sarin,Soman, Tabun, Temefos, Terbufos, Tetrachlorvinphos, Tribufos,Trichlorfon, Demecarium, Onchidal and the pharmaceutically saltsthereof.

In a more preferred embodiment, said acetylcholinesterase inhibitor is areversible acetylcholinesterase inhibitor chosen among: Aldicarb,Bendiocarb, Bufencarb, Carbaryl, Carbendazim, Carbetamide, Carbofuran,Chlorbufam, Chloropropham, Ethienocarb, Ethiofencarb, Fenobucarb,Fenoxycarb, Formetanate, Furadan, Ladostigil, Methiocarb, Methomyl,Miotine, Oxamyl, Phenmedipham, Pinmicarb, Pirimicarb, Propamocarb,Propham, Propoxur, Ganstigmine, Neostigmine, Phenserine and itsenantiomer Posiphen, Physostigmine, Pyridostigmine, Rivastigmine,eptastigmine (heptylphysostigmine), Acotiamide, Ambenonium, Donepezil,Edrophonium, Galantamine, its derivatives SPH 1371, SPH 1373, SPH 1375and SPH 1286 ((−)N-(3-piperidinopropyl)-N-demethylgalantamine),Huperzine A, its prodrug ZT 1((5R,9R)-5-(r-chloro-2-hydroxy-3-methoxybenzylidene-amino)-11-ethuidene-7-methyl-1,2,5,6,9,10-hexahydro-5,9-methanocycloocta[b]pyridin-2-one),Minaprine, Tacrine, tolserine(3,4,8b-trimethyl-2,3a-dihydro-1H-pyrrolo[2,3-b]indol-7-yl)N-(2methylphenyl) carbamate),(−)-12-amino-3-chloro-9-ethyl-6,7,10,11-tetrahydro-7,11-methanocycloocta[b]quinolinehydrochloride (huperzine X), Zanapezil and the pharmaceutically saltsthereof.

In another preferred embodiment, said acetylcholinesterase inhibitor ischosen among: ER 127528(1-(3-fluorobenzyl)-4-[(2-fluoro-5,6-dimethoxy-1-indanone-2-yl)methyl]piperidinehydrochlo-ride), thiatolserine, RS 1259 (N,N-dimethylcarbamic acid 4-[1(S)-(methylamino)-3-(4 nitrophenoxy)propyl]phenyl ester hemifumarate),ipidacrine (NIK-247), velnacrine(9-Amino-1,2,3,4-tetrahydro-1-acridinol), eptastigmine(heptylphysostigmine), zifrosilone(2,2,2-trifluoro-1-[3-(tri-methylsilyl)phenyl]ethanone), T 82(2-[2-(1-benzylpiperidin-4-yl)ethyl]-2,3-dihydro-9-methoxy-1H-pyrrolo[3,4-b]quinolin-1-onehemifumerate), CI 1002 (or PD 142676,1,3-dichloro-6,7,8,9,10,12-hexahydroazepino[2,1-b]-quinazoline), CHF2060 (N-heptylcarbamic acid2,4a,9-trimethyl-2,3,4,4a,9,9a-hexahydro-1,2-oxazino[6,5-b]indol-6-ylester-L-tartrate), MF 268(N-[8-(cis-2,6-dimethylmorpholin-4-yl)octyl]carbamic acid(3aS,8aR)-1,3a,8-trimethyl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indol-5-ylester L-bitartrate hydrate), TV 3326(N-propargyl-3R-aminoindan-5-yl-ethyl methyl carbamate) and Latrepirdine(Dimebolin) and the pharmaceutically salts thereof.

In a rather preferred embodiment, said acetylcholinesterase inhibitor isdonepezil, a reversible acetylcholinesterase inhibitor, or apharmaceutically salt thereof.

By “pharmaceutically acceptable salt” is meant for example a saltobtained by mineral or organic acid addition of basic residues such asamines; alkali or organic addition of acidic residues such as carboxylicacids and combinations comprising one or more of the foregoing salts.The pharmaceutically acceptable salts include non-toxic salts and thequaternary ammonium salts of the parent compound formed, for example,from non-toxic inorganic or organic acids. For example, non-toxic acidsalts include those derived from inorganic acids such as hydrochloric,hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; otheracceptable inorganic salts include metal salts such as sodium salt,potassium salt, cesium salt, and the like; and alkaline earth metalsalts, such as calcium salt, magnesium salt, and the like, andcombinations comprising one or more of the foregoing salts.Pharmaceutically acceptable organic salts includes salts prepared fromorganic acids such as acetic, trifluoroacetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,HOOC—(CH₂)_(n)—COOH where n is 0-4, and the like; organic amine saltssuch as triethylamine salt, pyridine salt, picoline salt, ethanolaminesalt, triethanolamine salt, dicyclohexylamine salt,N,N′-dibenzylethylenediamine salt, and the like; and amino acid saltssuch as arginate, asparginate, glutamate, and the like; and combinationscomprising one or more of the foregoing salts. Particular salts fordonepezil are disclosed in WO 2006/030249.

Donepezil(2,3-dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)-4-piperidinyl]methyl]-1H-i-nden-1-one)is a piperidine-based reversible, noncompetitive ChEI, which isindicated in the management of patients with Alzheimer's disease of mildto moderate severity.

Preliminary observations have suggested the value of donepezil in theamelioration of psychotic symptoms in patients with dementia of theAlzheimer's type (DAT), dementia with Lewy bodies and patients sufferingfrom Parkinson's disease (Bergman et al, Clin. Neuropharmacol. 2002;25(2):107-110, Birks J., Cochrane Database Syst Rev. 2006, (1):CD001190,Johanssen P. et al, CNS drugs 2006; 20(4):311-25, Gauthier S. et al,Curr. Med. Res. Opinion 2002; 18(6):347-54). Donepezil has been proposedfor treating numerous other cognitive disorders (vascular dementia,sleep apnea, mild cognitive impairment, schizophrenia, the CADASILsyndrome, attention deficit disorder, post coronary cognitiveimpairment, cognitive impairment associated with multiple sclerosis, andDown syndrome).

Although some clinical studies have been conclusive, the effect ofdonepezil is still debated, because the therapeutic effects are smalland are not always apparent. The clinical benefit of this cholinergicagent drug is in particular uncertain for patients at advanced stages ofthe disease for which the amplitude of the improvement is recognized aslimited (Nieoullon A., Psychol. Neuropsychiatr. Vieil. 2010;8(2):123-31). More importantly, some patients do not react to thesetreatments.

The present invention targets in particular a therapeutic substancecombination product containing at least one connexin-blocking agent andone acetylcholinesterase inhibitor (AChEI), wherein saidconnexin-blocking agent is meclofenamic acid and wherein saidacetylcholinesterase inhibitor (AChEI) is donepezil or a pharmaceuticalsalt thereof.

More precisely, the present invention targets a therapeutic substancecombination product containing at least one connexin-blocking agent andan acetylcholinesterase inhibitor (AChEI), as combination products forsimultaneous, separate or sequential use, as a medicament for treatingpatients suffering from cognitive disorders, wherein saidconnexin-blocking agent is meclofenamic acid and wherein saidacetylcholinesterase inhibitor (AChEI) is donepezil or a pharmaceuticalsalt thereof.

This combination product is for example a kit, containing, either in thesame recipient or in two distinct recipients, MFA and donepezil or apharmaceutical salt thereof.

The combination product of the invention contains preferably 1 μg/kg/dayto 1 mg/kg/day of donepezil or a pharmaceutical salt thereof. In apreferred embodiment, the combination product of the invention containsbetween 100 μg/kg/day and 1 mg/kg/day, and even more preferably between250 μg/kg/day and 1 mg/kg/day of donepezil or a pharmaceutical saltthereof. At these high doses, the combination with MFA enables toprolong the effect of donepezil during time, and to avoid secondaryeffects that often occur when such high doses are used.

In a preferred embodiment, the combination product of the inventioncontains between 1 μg/kg/day and 100 μg/kg/day, more preferably between10 μg/kg/day and 100 μg/kg/day, and even more preferably between 10μg/kg/day and 40 μg/kg/day of donepezil or a pharmaceutical saltthereof. At these low doses, the combination with MFA enables to obtaina sufficient effect of donepezil without triggering secondary effects.

The anti-inflammatory effect of MFA is observed in vivo at a dose ofapproximately 5 mg/kg/day (see Wagner C. et al, Am. J. Physiol. Regul.Integr. Comp. Physiol. 293 2007; R1781-6). However, when used in thecombination product of the invention, the amount of MFA is much lower,and typically comprised between 0.5 μg/kg/day and 0.5 mg/kg/day, morepreferably between 25 μg/kg/day and 0.5 mg/kg/day, and even morepreferably between 125 μg/kg/day and 0.5 mg/kg/day.

This combination product differs from the prior art in that, due to thevery low doses of MFA which are used, it does not affect theaccumulation of the Aβ proteins (Aβ₃₈, Aβ₄₀ or Aβ₄₂) and it does notinvolve the cyclooxygenase (COX) pathway. The use of MFA in thecombination of the invention is therefore not related at all with itsdescribed role as “non-steroid-anti-inflammatory drug” (NSAID), which isobserved at higher doses (typically above 5 mg/kg, see Wagner C. et al,Am. J. Physiol. Regul. Integr. Comp. Physiol. 293 2007; R1781-6).

Importantly, it has been demonstrated by the inventors that MFA,especially administrated at a low dose, potentiates donepezil and leadto an unexpected synergistic effect: although it has no effect on itsown at this low dose, MFA indeed enhances the effect of donepezil beyondwhat was thought to be its maximal effect (Bontempi B., et al,Neuropsychopharmacology 28 2003; 1235-46). Also, MFA accelerates thedonepezil's effect, which is observed as soon as 30 minutespost-treatment, while it is not expected before three-day post-treatmentwhen administered alone (see for example in Joo Y. et al, MolecularPharmacology 2006; 69:76-84).

As used herein, the term <<cognitive disorder>> means any conditioncharacterized by a deficit in mental activities associated withthinking, learning, or memory. Examples of such disorders includeagnosias, amnesias, aphasias, apraxias, deliriums, dementias, andlearning disorders. In some cases, the cause of a cognitive disorder maybe unknown or uncertain. In other cases, the cognitive disorder may beassociated with (that is, be caused by or occur in the presence of)other conditions characterized by damage to or loss of neurons or otherstructures involved in the transmission of signals between neurons.Hence, cognitive disorders may be associated with neurodegenerativediseases such as Alzheimer's disease, corticobasal degeneration,Creutzfeldt-Jacob disease, frontotemporal lobar degeneration,Huntington's disease, multiple sclerosis, normal pressure hydrocephalus,organic chronic brain syndrome, Parkinson's disease, Pick disease,progressive supranuclear palsy, or senile dementia (Alzheimer type); itmay be associated with trauma to the brain, such as that caused bychronic subdural hematoma, concussion, intracerebral hemorrhage, or withother injury to the brain, such as that caused by infection (e.g.,encephalitis, meningitis, septicemia) or drug intoxication or abuse; andmay be associated with Down syndrome and Fragile X syndrome.

Cognitive disorders may also be associated with other conditions whichimpair normal functioning of the central nervous system, includingpsychiatric disorders such as mild cognitive disorder, postcoronarybypass cognitive impairment, CADASIL syndrome, anxiety disorders,dissociative disorders, mood disorders, schizophrenia, and somatoformand factitious disorders; it may also be associated with conditions ofthe peripheral nervous system, such as chronic pain and neuropathicpain.

Examples of dementias are: AIDS dementia complex, Binswanger's disease,dementia with Lewy Bodies, frontotemporal dementia, multi-infarctdementia, Pick's disease, semantic dementia, senile dementia, sleepapnea in dementia, and vascular dementia. Examples of learning disordersare: Asperger's syndrome, attention deficit disorder, attention deficithyperactivity disorder, autism, childhood disintegrative disorder, andRett syndrome. Finally, examples of aphasia are progressive non-fluentaphasia.

In a preferred embodiment, the combination product of the inventionenables to treat the particular cognitive disorders chosen from:Alzheimer's disease, Parkinson's disease, vascular dementia and seniledementia.

According to another aspect, this invention also relates to the use ofthis combination product, simultaneously, separately or sequentially, inpatients suffering from cognitive disorders.

In the case of simultaneous use, the two components of the treatment areadministered to the patient simultaneously. According to this embodimentof the present invention, the two components can be packaged together,in the form of a mixture, or separately, then mixed spontaneously beforebeing administered together to the patient. More commonly, the twocomponents are administered simultaneously, but separately. They can forexample be administered with an interval of time which is typicallycomprised between few minutes and several hours, preferably between 1minute and five hours, more preferably between 1 minute and two hours.

In particular, the routes of administration of the two components may bedifferent. The administration can also be performed at different sites.In another embodiment, the two components are administered sequentiallyor spaced apart over time, for example in the same day or at an intervalranging from several hours to several weeks, or even several months.

The present invention also involves the use of at least oneconnexin-blocking agent such as MFA for preparing a drug intended to beadministered before, at the same time, or after an acetylcholinesteraseinhibitor, such as donepezil, in order to treat a patient suffering fromcognitive disorders.

According to another aspect, the invention includes the use of at leastone connexin-blocking agent such as MFA, for potentiating the effect ofan acetylcholinesterase inhibitor such as donepezil in patientssuffering from cognitive disorders.

The present invention also targets the use of at least oneconnexin-blocking agent such as MFA, for potentiating the effect of anacetylcholinesterase inhibitor such as donepezil in patients sufferingfrom cognitive disorders.

According to another aspect, the invention thus includes aconnexin-blocking agent such as MFA for use to potentiate the effect ofan acetylcholinesterase inhibitor such as donepezil in patientssuffering from cognitive disorders. All the embodiments concerning theacetylcholinesterase inhibitor, the connexin-blocking agent and thecognitive disorders are hereby encompassed.

The present invention also targets a method for potentiating the effectof an acetylcholinesterase inhibitor such as donepezil, in patientssuffering from cognitive disorders, said method using at least oneconnexin-blocking agent, for example MFA.

The term “potentiate” in this case means significantly increasing theeffects of the cholinergic agent administered before, simultaneously orafter the anti-connexin agent. In particular, the combination of thecholinergic agent with the anti-connexin agent makes it possible toenhance the therapeutic effect of said cholinergic agent, to an extendwhich is higher than that obtained by the cholinergic agent alone,whatever its concentration is considered. A “significant increase” ofthe effects of the cholinergic agent is obtained for example when thiseffect is enhanced by at least about 25%, preferably at least about 40%and more preferably by at least about 50% as compared with the effect ofthe cholinergic agent alone. Said effect can be for example measured byanalyzing the EEG profile or the percentage of alternation relative tovehicles in laboratory animals as described in the experimental partbelow.

This enhanced effect (“potentiation”) also enables to reduce the dosesat which said cholinergic agent is used, and therefore to limit thepotential adverse effects of said cholinergic agent, and/or to reducethe effects of failure and withdrawal.

The invention therefore also relates to the use of at least oneconnexin-blocking agent, for reducing the doses of said cholinergicagent and/or limiting the adverse effects of said cholinergic agent,and/or reducing the effects of failure and withdrawal.

Donepezil is typically used at a dose comprised between 5 mg to 10 mgper day for an adult individual, which means approximately between 100μg/kg/day and 200 μg/kg/day. As disclosed above, the combination of theinvention enables either to reduce the said doses, typically to lessthan 100 μg/kg/day and preferably to less than 50 μg/kg/day (forexample, the dose of donepezil can be comprised between 1 μg/kg/day and100 μg/kg/day, preferably between 10 μg/kg/day and 100 μg/kg/day, andmore preferably between 10 μg/kg/day and 40 μg/kg/day) or to maximizethe effect of donepezil over time without inducing secondary effects(when donepezil is used at higher doses, that is, between 100 μg/kg/dayand 1 mg/kg/day, preferably between 100 μg/kg/day and 200 μg/kg/day, orbetween 250 μg/kg/day and 1 mg/kg/day).

According to a final aspect, the invention describes a method fortreating a patient suffering from cognitive disorders, including theadministration to said patient of:

a) at least one acetlycholinesterase inhibitor, and

b) at least one connexin-blocking agent,

and in which said products a) and b) are administered simultaneously,separately or spread out over time.

All embodiments of this method are as described above.

Examples 1. Materials and Methods

1.1. Electro-Encephalographic Recording in Mice.

Electrophysiological effects of donepezil were evaluated by analysis ofhippocampal electroencephalographic activity (EEG) as describedpreviously in WO2010/029131.Briefly, the assay is as follows:

Pre-Implantation of the Electrodes:

Two groups of male C57bl/6 mice (seven mice from 4 to 5 month old andseven mice from 17 to 18 month old) were pre-implanted with bilateralhippocampal bipolar electrodes under isoflurane anaesthesia. A two-weekperiod of recovery was realized before recordings.

Injections:

Different intraperitoneal treatments were performed by circularcombination of 7 mice per treatment (donepezil 0.1 and 0.3 mg/kg,meclofenamic 1 mg/kg, donepezil 0.1 mg/kg+MFA 1 mg/kg). The dose of 1mg/kg of MFA has previously been described as not affecting theelectroencephalographic signal in rodents.

EEG Measures:

EEG measures were performed on different batches of awoke mice(previously implanted and tamed) by recordings two hours afterinjection. The spectral analysis is carried out by Fourier transform(FFT) and allows the calculation of the relative powers for each Hertzand each second. FFT data are then averaged minute by minute andreported to control solvent recording realized on the day before instrictly identical experimental conditions. The spectral powers of twohippocampal electrodes are then averaged between 3 and 12 Hertz andrepresented hourly.

At day 1, saline was administered intraperitoneally to “adult” or“elderly” mice (n=7), and θ-hippocampal activity was measured for twoconsecutive hours. On day 2, donepezil alone or in combination with MFAis injected, and θ-hippocampal activity is related to that measured onday 1. Results are shown on FIG. 1 (*: p<0.05 (One-way ANOVA).

1.2. Behavioral Test of Working Memory—T-Maze Protocol in Mice

The alternating sequential test is widely used to assess spatial workingmemory in mice (Beracochea D. J. and Jaffard R., Behav. Neurosci. 101(1987) 187-97). Spontaneous alternation is the innate tendency ofrodents to alternate their choices to enter into the compartments ofarrival of a T-maze device, over successive trials. To alternate duringa given trial N, the animal must remember the choice made selectively intest N−1, so the decline in alternating will reflect the phenomenon ofoblivion. The response in alternating is performance measure. Sequentialalternating assess more specifically the sensitivity to interference, amajor factor in oblivion.

The experiment took place in a T-maze (50 cm×10 cm×25 cm). All thesubjects (C57bl/6 male mice, 17-18-month old, n=9) were given 7successive trials separated by a 90-s intertrial interval. To begin atrial, the mouse was placed in the start box for 90 s before the door tothe stem was opened. When the subject entered one of the goal arms, thedoor to that arm was closed. The chosen arm and the time that elapsedbetween opening the door and the arrival to the end of the chosen arm(task achievement time) were registered. Following a 30-s confinementperiod in the chosen arm, the animal was removed and placed in the startbox for a new trial. Between each test, the unit was cleaned with waterand alcohol to avoid olfactory detection. An alternation response wasconsidered each time the subject entered the arm opposite to the onevisited on the immediately previous trial. Alternation rate wascalculated taking into account the 6 successive trials, and expressed inpercentage relative to the maximal alternation rate of 100% (obtainedwhen the subject never returned into the same arm for two consecutivetrials).

C57BL/6 mice of 17 to 18 months (“elderly” mice) were intraperitoneallyinjected with a solution of NaCl (vehicle), donepezil (DZP),meclofenamic acid (MFA) or a combination of these two latter compounds,30 minutes before T-maze experiment.

Thirty minutes after treatment (NaCl, donepezil, MFA), “elderly” miceare placed in the T-maze device. The percentage of alternation wasmeasured for 7 consecutive trials, 50% corresponding to a randomalternation. Results are shown on FIGS. 2 and 3 (**: p<0.01; *: p<0.05(ANOVA)).

1.3. Statistic Analysis

Statistical analysis was established by SigmaPlot software (SystatSoftware Inc).

2. Experimental Results 2.1. Study of Donepezil Potentiation byElectroencephalography

As it is known that increased electrical activity in the CNS, measuredon an electroencephalogram (EEG) reflects in some circumstances thetherapeutic benefits of a psychoactive drug (Galderisi S. et al, MethodsFind. Exp. Clin. Pharmacol. 24 Suppl D (2002) 79), the effect of thecombination donepezil/MFA was evaluated on θ-hippocampal activity fortwo hours and on two groups of mice (“adult” and “elderly”) as shown inFIG. 1.It has been observed that:

-   -   4-5-month old mice do not respond significantly to the        administration of donepezil 0.1 mg/kg and 0.3 mg/kg, while        recordings of 17-18-month old mouse show during the second hour        of recording, an effect of donepezil at 0.3 mg/kg. This is        consistent with the different responses to donepezil described        between “adult” and “elderly” mice (Tronche C. et al, Behav.        Brain. Res. 2010; 215:255-260).    -   Meclofenamic acid potentiates significantly, during the first        and the second hour, the pharmacological effect of donepezil.        Accordingly, EEG showed that, whereas MFA had no effect by its        own, the combined treatment with donepezil was more potent than        donepezil alone. As revealed by ANOVAs, MFA+donepezil increased        theta frequency by more than 50% compared to vehicle and        donepezil (0.1 and 0.3 mg/kg) treated mice (n=7 per group;        p=0.034).

2.2. Study of Donepezil Potentiation by Behavioral Analysis

Donepezil is a promnesiant molecule described as improving theperformance of mice in T-maze devices (Spowart-Manning L., Behav. Brain.Res. 151 (2004) 37-46).

It can be inferred from FIG. 2 that:

-   -   Donepezil has a significant promnesiant effect a 0.3 and 1        mg/kg, identified by an increase of alternation due to a recall        memory of the choice made in the previous test. This molecule        has no significant promnesiant effect 0.1 mg/kg.    -   Meclofenamic acid has no promnesiant effect significant at 1        mg/kg.    -   Donepezil at 0.1 mg/kg is potentiated by meclofenamic acid        The alternation of mice was also analysed with blocks of two        trials (Block 1: trials 2 and 3/Block 2: trials 4 and 5/Block 3:        trials 6 and 7). The results are shown in FIG. 3:    -   Meclofenamic acid shows no significant effect on the three        blocks of trials.    -   The performance of control mice (“vehicle”) are degraded in the        third block, reflecting the effect of memory interference.    -   Donepezil at 0.1 mg/kg partially counteract the interference        phenomenon in the third block of trials.    -   Meclofenamic acid mainly potentiates the effect of donepezil in        the third block, and enhances the recall of memory by reducing        the interference phenomenon.

More precisely, the combination with meclofenamic acid allows to reach ahigher effect (almost 85%) than the maximal effect observed fordonepezil alone, and for a lesser dose of donepezil (0.1 mg/kg, see FIG.2, last column). In other words, the combined treatment of MFA+donepezilat 0.1 mg/kg was revealed more efficient than highest doses of donepezilalone (i.e. 0.3 and 1 mg/kg) to reverse the age-induced impairment (n=9per group; p<0.01).

Also, the combination with meclofenamic acid allows to obtain a highermnesic interference resistance than the one induced by donepezil. Thisimprovement of the effect of donepezil has never been observed so far.

These results are surprising. As a matter of fact, as it can be seen inliterature (cf. for example Bontempi B. et al (Neuropsychopharmacology2003; 28; 1235-12460) for the dose of 0.2 mg/kg s.c.,) or in theexperiments shown above (0.3 mg/kg i.p.), donepezil shows its maximalefficiency at 0.2-0.3 mg/kg (see FIG. 2: around 75% obtained for 0.3mg/kg and 1 mg/kg of donepezil alone). Higher efficiency was notachievable with donepezil alone, even at higher doses.

OVERALL CONCLUSIONS

Table 1 below resumes the electroencephalographic (EEG) and behavioural(SA-task) results.

donepezil donepezil donepezil 0.1 mg/kg + Statistics 0.1 mg/kg 0.3 mg/kgMFA 1 mg/kg (ANOVAs) EEG 1 1 1.6* p = 0.034  Relative power to vehiclesSA-task relative 35% 48%** 68%** p = 0.0013 to vehicles

EEG results are expressed as the relative power of hippocampal thetarhythm of each group compared to vehicles injected mice. SA-task resultsare expressed as the mean percentage of alternation relative to vehiclesover the seven trials. *: p<0.05; **: p<0.01.

It has been shown for the first time that the electrophysiologicalactivity and promnesiant activity of donepezil is greatly potentiated bymeclofenamic acid, which surprisingly allows a maximal pharmacologicaleffect of donepezil.

In addition, meclofenamic acid modifies the pharmacological profile ofdonepezil, modifying the temporal evolution and intensity of its effectson memory.

It is important to note that MFA, during acute treatment, has no owneffect on the preclinical model used herein, so that MFA's activity isthought to be independent from Aβ accumulation:

Independence Between Aβ and NSAID Activity.

The preclinical model used herein (middle-aged wild-type 17-18 monthmice) is a pathological model of cognitive impairment which is notcharacterized by an Aβ accumulation. Indeed, there is no Aβ accumulationin wild-type mice, even beyond 24 months (see FIG. 2 in Walther T. etal, PLoS One 2009; e4590), although a cognitive decline is depicted at17-18 months (see in Beracochea D. et al, Psychopharmacology (Berl) 1932007; 63-73). In addition, no cognitive significant effect of MFA isseen at 1 mg/kg alone, (FIGS. 2 and 3 of this application) while suchdose potentiate donepezil.

For its anti-Cox activity MFA is administrated at dose above 5 mg/kg(see in Wagner C. et al, Am. J. Physiol. Regul. Integr. Comp. Physiol.293 2007; R1781-6). However, MFA was used in the present invention at adose lower than those published for its NSAID activity.

This point constitutes a major difference with prior art documents suchas Mc Gleenon et al, British Journal of Clinical Pharmacology, 1999; 48,471-480, and Gasparini L. et al, Journal of Neurochemistry 2004; 91,521-536, since this latter document clearly states that Aβ doses aresimilar to anti-Cox doses. Thus, the potentiating effect of MFA ondonepezil is independent from its potential anti-Aβ₁₋₄₀ ou anti-Aβ₁₋₄₂effects.

-   -   The present invention does not specifically target Alzheimer's        disease but all cognitive impairments.        -   The present invention broadly addresses all cognitive            disorders. Such disorders are qualified by the alteration of            specific cognitive functions such as attention, memory,            language, seen in numerous dementias (Alzheimer's disease,            Lewy-body dementia, dementia associated with Parkinson's            disease, senile dementia, vascular dementia, hydrocephalia,            Korsakoff syndrome, Creutzfeldt-Jakob disease, etc.). Thus,            the present invention does not specifically target a disease            with Aβ accumulation but rather all types of cognitive            impairment, independently from their etiology (as a matter            of fact, donepezil is also described at targeting            Aβ-independent mechanisms such as the cholinergic system).    -   MFA, administrated—at a low dose and in the above-exemplified        preclinical model—potentiates donepezil, which is an unexpected        effect:        -   This effect is not an additive effect but an unexpected            synergistic one. As a matter of fact, MFA has no own effect            at the tested dose, and donepezil is potentiated beyond its            maximal effect (Bontempi B., et al, Neuropsychopharmacology            28 2003; 1235-46) (notably with respect to resistance to            memory interference, which is altered in human dementia            (Hanseeuw B. J. et al, Brain Cogn. 72 325-31).        -   The kinetics of the activity of the combination of the            invention is new and unexpected. As a matter of fact, the            potentiation of donepezil's efficiency is observed as soon            as 30 minutes post-treatment, while effects described in            prior art documents (such as Joo Y. et al, Molecular            Pharmacology 2006; 69:76-84) are only seen after a three-day            treatment.

1. Therapeutic substance combination product containing at least oneconnexin-blocking agent and one acetylcholinesterase inhibitor (AChEI),wherein said connexin-blocking agent is meclofenamic acid and saidacetylcholinesterase inhibitor (AChEI) is donepezil or a pharmaceuticalsalt thereof.
 2. The combination product according to claim 1,containing between 0.5 μg/kg/day and 0.5 mg/kg/day of meclofenamic acid.3. The combination product according to claim 1, containing between 1μg/kg/day and 1 mg/kg/day of donepezil or a pharmaceutical salt thereof.4. Method of treating cognitive disorders, which comprises administeringto a patient in need there of an effective amount of a therapeuticsubstance combination product containing at least one connexin-blockingagent and an acetylcholinesterase inhibitor (AChEI), wherein saidconnexin-blocking agent is meclofenamic acid and saidacetylcholinesterase inhibitor (AChEI) is donepezil or a pharmaceuticalsalt thereof, and wherein said connexin-blocking agent and saidacetylcholinesterase are administered simultaneously, separately orsequentially.
 5. The method according to claim 4, wherein said cognitivedisorder is chosen from Alzheimer's disease, Parkinson disease, vasculardementia and senile dementia.
 6. The method according to claim 4,wherein the therapeutic substance combination product contains between0.5 μg/kg/day and 0.5 mg/kg/day of meclofenamic acid.
 7. The methodaccording to claim 4, wherein the therapeutic substance combinationproduct contains between 1 μg/kg/day and 1 mg/kg/day of donepezil or apharmaceutical salt thereof.
 8. Method of potentiating the effect ofdonepezil or a pharmaceutical salt thereof in patients suffering fromcognitive disorders, which comprises administering to said patient aconnexin-blocking agent wherein said connexin-blocking agent ismeclofenamic acid.
 9. The method according to claim 8, wherein saidcognitive disorder is chosen from Alzheimer's disease, Parkinsondisease, vascular dementia and senile dementia.
 10. The method accordingto claim 8, wherein the meclofenamic acid is used at a dose between 0.5μg/kg/day and 0.5 mg/kg/day.
 11. The method according to claim 8,wherein the donepezil or a salt thereof is used at a dose between 1μg/kg/day and 1 mg/kg/day.
 12. The combination product according toclaim 2, containing between 1 μg/kg/day and 1 mg/kg/day of donepezil ora pharmaceutical salt thereof.
 13. The method according to claim 5,wherein the therapeutic substance combination product contains between0.5 μg/kg/day and 0.5 mg/kg/day of meclofenamic acid.
 14. The methodaccording to claim 5, wherein the therapeutic substance combinationproduct contains between 1 μg/kg/day and 1 mg/kg/day of donepezil or apharmaceutical salt thereof.
 15. The method according to claim 6,wherein the therapeutic substance combination product contains between 1μg/kg/day and 1 mg/kg/day of donepezil or a pharmaceutical salt thereof.16. The method according to claim 9, wherein meclofenamic acid is usedat a dose between 0.5 μg/kg/day and 0.5 mg/kg/day.
 17. The methodaccording to claim 9, wherein donepezil or a salt thereof is used at adose between 1 μg/kg/day and 1 mg/kg/day.
 18. The method according toclaim 10, wherein donepezil or a salt thereof is used at a dose between1 μg/kg/day and 1 mg/kg/day.